Acidity as measured by pH affects the rate and extent of chemical reactions. Measurement of pH of biological fluids is important for following the health of an organism. Furthermore, its measurement in individual cells and tissues has been correlated with response to external stimuli including drugs, ions, light and other cells. Acidity is also important in many non-biological systems and a variety of electrical and spectroscopic techniques have been developed for its measurement. Measurement of pH by optical sensors is an established technique. Absorption indicator dyes such as phenolphthalein have been used for visual or instrumental estimation of acidity for many years. The basic requirement for measurement with an optical indicator is a change in an optical property of the dye such as absorption or fluorescence that can be correlated with the pH of the medium. For the greatest sensitivity to small changes in pH of the medium, the equilibrium constant between the acidic and basic forms of the indicator for the dye (i.e. the pK.sub.a) should be near the pH of the medium. For measurements in physiological media including blood and the interior of most cells, this is usually in the range of pH 6 to pH 8, commonly pH 7.0 to 7.6. An advantage of measurement of pH by a fluorescence technique rather than absorbance is the greater sensitivity of the measurement due to the intrinsically higher detectability of emitted light versus absorbed light. The correspondingly lower levels of sensor required permit measurements of the intracellular pH in single cells by such techniques as flow cytometry with concentrations of the sensor that have low toxicity and avoid buffering of the equilibrium.
Among the fluorescent dyes that have been used to estimate pH, the most common are .beta.-methylumbelliferone, its derivatives, 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine)(Wolfbeis, et al., Z. Anal. Chem. 314, 119 (1983)), and fluorescein or its derivatives, carboxyfluorescein (Thomas, et al., Biochemistry 18, 314, 119 (1979)), and 2',7'-bis-(carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) (Rink et al., J. Cell. Biol. 95, 189 (1982)). Of these, only the fluorescein dyes have the long wavelength spectral properties comparable to the dyes in this invention. This includes absorbance at the primary wavelengths of the argon laser (488 and 514 nm). This laser is widely used in flow cytometry and fiber optic monitoring. Fluoresceins have pH dependent absorption and excitation spectrum and a pK.sub.a of approximately 6.4 to 7.0 depending on substituents. Measurement of pH dependent emission intensity changes in single cells with a single excitation wavelength gives spurious results since the intensity will be effected by dye concentration in the cell, by leakage of the dye out of the cell, cell thickness, and photobleaching of the dye. Measurement of pH by the emission intensity with a single excitation wavelength by use of a dye immobilized on a fiber optic suffers similar problems related to dye concentration, bleaching and path length. A more practical approach that reduces the effect of changes in dye concentration in the light path is to ratio the amount of fluorescence at a fixed wavelength with excitation at a pH sensitive wavelength to the amount of fluorescence at the same wavelength with excitation at a relatively pH insensitive wavelength. This is the technique commonly used to estimate the pH inside cells with fluorescein derivatives such as BCECF (Paradiso, et al., Nature 325, 477 (1987)). While this is quite practical for suspensions of cells and in homogeneous fluids in a fluorometer or a microscope, because measurement of pH by ratioing excitations requires excitation sources of two different wavelengths, this is usually impractical in flow systems including flow cytometers and fiber optic systems for continuous monitoring of the pH of flowing fluids, such as blood.
For the reasons outlined above, it is preferable in a flowing system to be able to excite the sensing dye at a single wavelength and to be able to monitor fluorescence emission at two wavelengths with the maximum emission in acidic solution at a different wavelength than in basic solution. Furthermore, it is strongly advantageous if the dye possesses excitation and emission wavelengths above 480 NM both to take advantage of current laser excitation sources and, in the case of biological fluids, to decrease the background from natural fluorescence in cells and fluids and to reduce the intrinsic light scattering artifacts (which decrease with the inverse fourth power of the wavelength). Only the dyes described in this invention have been reported to possess this and other desirable properties. In the pH range 5 to 9, emission from fluorescein and related derivatives is all from the base form with wavelengths that are essentially independent of the pH of the medium. This has been explained as being due to the higher acidity of the excited state relative to the ground state leading to emission from a single state. While the population of the excited state may be altered by a quenching of the absorbance by a change in pH of the medium, the fluorescence emission spectra for fluorescein-based dyes over the pH range 5 to 10 changes only in intensity and not in wavelength thus precluding ratioing of emission wavelengths (Graber, et al., Analyt. Biochem. 156, 202 (1986)).
Dihydroxyphthalonitrile (U.S. Pat. No. 4,503,148) shows pH dependent emission but suffers from a complex response to pH indicative of multiple equilibria and, since it has only relatively weak ultraviolet and near UV absorbance, cannot be excited with the principal wavelengths of the argon laser (488 nm and 514 nm) in common use in flow cytometry. Unlike the benzo[c]xanthene dyes that are the subject of this invention, the pH of the medium as indicated by this dye is not readily determinable by ratioing emission intensities. Furthermore, it cannot be directly immobilized on materials such as optical fibers without modification of its fluorescence properties and, due to its high membrane permeability, is not well retained by cells.